Article Figures & Data
Figures
- FIGURE 1.
Both DPI and GSK inhibit superoxide release from human neutrophils as measured by cytochrome c reduction.
Shown is a representative experiment (A) with cells preincubated for 5 min with buffer (KRG) or 27 μM indicated inhibitor before stimulation with PMA (50 nM; arrow), and a dose–response graph (B) displaying PMA-triggered superoxide release (as compared with samples in the absence of inhibitor) in the presence of indicated concentrations of DPI (triangles) or GSK (circles) as mean ± SD of three independent experiments. Negative control values obtained from PMA-stimulated samples in the presence of SOD were subtracted, and the 100% response value (no inhibitor) was set to x values of 10−9 M to be able to present the data on a logarithmic axis. Statistical differences between GSK and DPI are indicated. (C) Neither inhibitor caused general cell damage, as cells treated with high concentrations of inhibitors (40 μM) were perfectly capable of intracellular calcium signaling in response to chemoattractant stimulation. Representative calcium transients (out of at least three independent experiments) are shown, and arrows indicate the time points for addition of fMLF (10 nM). ***p < 0.001.
- FIGURE 2.
ROS production at distinct sites.
PMA stimulation of human neutrophils results in ROS production at two distinct sites, extracellular and intracellular, that can be measured independently by isoluminol- and luminol-enhanced CL, respectively (A). High doses (10 μM) of either GSK or DPI blocked EC as well as IC ROS production (B). Representative (out of at least 10 independent experiments) curves are shown (left), and bar diagrams (right) compare peak values (mean + SD; n = 3) after PMA stimulation in the presence or absence of inhibitors (10 μM), with statistical significance (using paired Student t tests) for each inhibitor as compared with controls with KRG buffer instead of inhibitor. Cells were incubated with inhibitors for 5 min at 37°C before stimulation with PMA. EC, extracellular; IC, intracellular. ***p < 0.001.
- FIGURE 3.
Inhibition of extracellular and intracellular ROS production induced by PMA.
Dose–response curves for inhibition of PMA-stimulated extracellular versus intracellular ROS production by GSK (A) or DPI (B). The 100% response value (no inhibitor) was set to x values of 10−10 M to be able to present the data on a logarithmic axis. Cells were incubated with inhibitors for 5 min at 37°C before stimulation with PMA. Shown are mean ROS peak values as percentages of control ± SD of three independent experiments performed in triplicate. Statistically significant differences between the EC and IC responses are indicated for each inhibitor dose. EC, extracellular; IC, intracellular. **p < 0.01, ***p < 0.001.
- FIGURE 4.
Inhibition of extracellular and intracellular ROS production induced by fMLF or phagocytosis.
Dose–response curves for inhibition of fMLF-stimulated extracellular versus S. aureus–triggered intracellular ROS production by DPI (A). Cells were incubated with inhibitor for 5 min at 37°C before stimulation. Shown are mean ROS peak values as percentages of control ± SEM of three independent experiments in triplicate. Statistically significant differences between the EC and IC responses are indicated for each inhibitor dose. The 100% response value (no inhibitor) was set to x values of 10−10 M to be able to present the data on a logarithmic axis. (B) DPI (10 μM) did not decrease MPO activity in neutrophil lysates. Shown are mean + SD of three independent experiments. EC, extracellular; IC, intracellular. ***p < 0.001.
- FIGURE 5.
Kinetics of NADPH oxidase inhibition.
Kinetics of the inhibitory effects of DPI (A) and GSK (B) on the extracellular (solid lines) and intracellular (dotted lines) NADPH oxidase. Each inhibitor (20 μM) was injected (arrows) during PMA-triggered ROS production, and the CL responses were followed; note that x-axes display s. Shown are representative curves out of at least four independent experiments. EC, extracellular; IC, intracellular.
- FIGURE 6.
Reversibility of ROS production.
Reversibility of ROS production after washing away GSK (black) or DPI (white). (A) Cells were incubated with each inhibitor (20 μM) for 5 min and then washed three times with inhibitor-free buffer before being assayed for extracellular (left) and intracellular (right) ROS production in response to PMA. Shown are mean + SD of ROS peak values obtained from four independent experiments. The response of control cells incubated with buffer (KRG) before being subjected to the same washing procedure and PMA stimulation is set to 100%. Statistical significance between GSK and DPI was calculated by paired Student t tests. (B) That the inhibitory effect of DPI on extracellular ROS production could be washed away was ascertained by cytochrome c reduction assay. Cells were incubated with DPI (20 μM) for 5 min and then washed and stimulated with PMA in the presence or absence of new DPI (20 μM). The graph displays mean Δ values (max OD − initial OD) + SD from two independent experiments performed in triplicate. Statistical significance was calculated by an unpaired t test. *p < 0.05, ***p < 0.001.
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